Exploring Molecular Dynamics: FRET and Confocal Microscopy Techniques

This presentation provides a comprehensive overview of Fluorescence Resonance Energy Transfer (FRET) and Confocal Microscopy, two crucial techniques in biophysics and biochemistry. It details the principles of FRET, emphasizing its sensitivity to molecular distances and its use in analyzing molecular kinetics, protein interactions, and conformational changes. The presentation also covers the application of single-pair FRET (spFRET) in studying enzyme behavior, specifically focusing on H+-ATP synthases, and how these methods are used to track subunit movements during catalysis. Furthermore, the presentation explains the working principles of confocal microscopy, including its three-dimensional imaging capabilities and the suppression of out-of-focus signals. It describes the experimental setup, including laser systems, dichroic mirrors, and pinholes, used to detect molecular movements. The presentation concludes by discussing the limitations of both FRET and confocal microscopy, such as environmental sensitivity and laser irradiation effects. Finally, the presentation references a study that applied FRET and confocal microscopy to study domain movements during the catalytic reaction process.

Slide 1 Cover page
Slide 2 FRET
● The effectiveness of energy transfer in this process is highly depended on the distance
between the two molecules, which results in high sensitivity of the molecules towards the
changes in the distance between the two molecules under consideration .
● FRET is a useful methodology to determine molecular kinetics and dynamics in the field
of biophysics and biochemistry. The molecular interaction which is monitored using
FRET is protein interaction with the protein, interaction of protein with the
deoxyribonucleic acid (DNA) and change in the protein conformational structure.
Side 3
AIM
● In order to measure the domain movement using this technology, two fluorophores were
attached at the appropriate cite.
● To analyze the subunit movement, one site for the attachment of chromosphere was
rotor subunit and the other was attached at stator subunit.
Slide 4
Single-pair fluorescence resonance energy transfer (spFRET)
Blue color ribbon represents rotor subunit and orange color is associated with stator unit.
The donor location e56C is displayed in green and the acceptor location b64C is presented in red.
● Binding sites for the attachment of fluorophores was selected based on the homology
model, and it was observed that b subunit was more suitable for the purpose of labeling.
● The homology model described the distance among cysteine residues on the rotor and
stator, that was about 4 and 8 nm respectively.
● In the present study spFRET applied as an analysis tool analyze the interaction of enzyme
“H+-ATP synthases” from bacteria and chloroplast from the spinach at the time of
catalysis. It was applied to track the movement of enzymes and to analyze the kinetics at
which subunits changes its place at the time of catalysis process. It was observed that the
synthesis of ATP was at 50 ms and it should correspond to the domain movement in the
similar time period . FRET efficiency was calculated and it was observed that FRET
efficiency was independent of the enzyme position in the confocal volume. However, it
was highly dependent on the distance between the donor and acceptor molecules along
with their orientation.

Slide 5
To remove the problem associated with multiple acceptor and donor site Cy5bis dye was
used, which assisted in the crosslinking of two b subunits, which provides a defined stalk
position.
The FoF1 tagged with the acceptor fluorophore was introduced to the membrane of liposome
followed by the removal of F1.
The final step involving the labeling part is the attachment of F1 previously tagged with an
donor fluorophore to the membrane.
The overall process provides a proteoliposmome, that have a donor and acceptor attached
together in a single subunit.
Limitation of FRET
● FRET is highly sensitive to the change in local environment.
● FRET uses different fluorescence label, which can individually different sensitivity to
different environmental factor.
Slide 6
● The process of targeting light or laser on a specific material resulting into the generation
of lights of different color from the particle is known as fluorescence.
● The atoms or molecules present in the particle reaches to the high energy level known as
the excitation of the molecule.
● As the molecule returns to the ground state it emits photons based on the excitation
energy absorbed by the molecules. The photon energy determines the color of light
emitted from the particle of interest .
● It is a technique which provides a three-dimensional (3D) imaginary of a biomolecule in
the form an optical resolution. The 3D resolution is gathered by formulating the
parameters of the instrument in order to calibrate instrument sensitivity towards the
particle of interest in a focal plane.
● The working principle of confocal microscopy depends on the suppression of the extra
signals or the noise which is emerging from the planes in the out of the focal region. The
suppression is achieved with the help of pinhole formation prior to the detection unit. The
emerging from the focal plane passes freely from the pinhole and the out of focal plan
light is mostly hindered and stopped by the pinhole.

● The particle of interest in the confocal fluorescence microscopy is generally targeted by
the laser which results into the illumination of the particle or biomolecule. The
illumination results into the generation of contrast with respect to other the particles in
the close proximity of the particle of the interest .
Slide 7
● The detection of movement of F1F0 domain movement during the catalytic reaction
process was carried out using confocal microscopy.
● The setup for the detection was comprised of Nd:YAGA laser, telescope, an objective
lens immersed in the water, dichroic mirror and a pinhole (100 μm).
● The general working involved in the detection purpose is as follows, the beam of laser
light is focused on the sample of interest in the present case labeled domain, the focus is
made with the help of a telescope, through a objective which is immersed in the water at
the centre point of the sample.
● The sample produces fluorescence due to the labeled fluorophores molecules attached to
the each of the samples.
● The fluorescence from the material of interest was collected by the objective, further
passes through the dichroic mirror.
● The superimposition created by the Gaussian intensity distribution produced by the laser
and ellipsoidal focus of the pinhole, which is presented in yellow in the figure, makes a
three-dimensional confocal volume.
● The confocal volume could be varied between 10 fL to 100 fL with the help of a pin hole.
The confocal volume was determined by the FCS, using rhodamine 6G for
standardization.
Slide 8
• In the setup for confocal microscopy, the fluorescence was separated in two different
wave length based on the photon intensity of donor and accepter ligand.
• The donor wavelength was in the range of 540 nm to 610 nm.
• The acceptor wavelength was in the range of 665 nm to 740 nm.
• The separation of the wavelength was carried out using a second dichromic filter and a
interface filter.
• The interface filter for the donor region was HQ575/65, in case of the acceptor region it
was 665lp.

Slide 9
In the present study the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser of 532
nanometer wavelength was reduced to 100 μW and the beam was focused with the help of
telescope along with a water immersed objective piece. The photons were detected using
photodiodes.
Limitation of confocal microscopy
Limitation of Confocal microscopy involves, the limited number of excitation wave length
involved which provides a narrow wave length. The harmful aspect of high intensity laser
irradiation to the living cell and tissue is one more limitation in the use of confocal microscope.
Slide 10
References
Bienert, , Zimmermann, B., Rombach‐Riegraf, V. & Gräber, , 2011. Time‐Dependent FRET with
Single Enzymes: Domain Motions and Catalysis in H+‐ATP Synthases. ChemPhysChem,
pp.510-17.